Liquid chromatography (in particular HPLC) is used for the purpose of separating liquid samples into their components by means of a chromatography column. In this case, the separating power of the (separating) column is dependent, inter alio, on its length and on the particle size of the packing material. For the best possible separation, columns having a sufficient length and a small particle size are required. Such columns have a high flow resistance and therefore require substantially higher pressures for operation than conventional columns.
Furthermore, a sufficiently rapid separation is desirable, to enable a high sample throughput. This requires a high flow speed in the column, whereby the counterpressure in the column also increases.
One possibility for accelerating the separation or increasing the separating power is to change the solvent composition over the duration of the separation (referred to hereafter as the gradient, i.e., the degree of the change of the elution force or the proportion of a solvent with time is referred to as the gradient or solvent gradient).
Two different technical implementations for achieving a gradient have prevailed due to the different requirements for the separation. These are so-called high-pressure gradient forming (HPG) and low-pressure gradient forming (LPG). High-pressure gradient forming operates by means of two independent pumps, which are connected to one another via a T-part on the high-pressure side of the system. The gradient is generated by way of the change of the flow rates at the two pumps. Low-pressure gradient forming only requires one pump having proportioning valve unit connected upstream. During the aspirating cycle of the pump, the various solvents are drawn successively into the pump by opening and closing the valves (for example, solenoid valves) in the proportioning valve unit. The gradient is formed due to the variations in the opening times for the various solvents. To smooth out composition fluctuations in the solvent mixture (mixture irregularity), a mixer is connected downstream from the pump as a standard feature.
In the various designs, there are different so-called gradient delay volumes (GDV—or also dwell volume). The gradient delay volume or mixing volume is dimensioned, on the one hand, by way of the holding capacity of all interconnected components from the mixing point up to the entry of the column. In the case of LPG, the GDV—depending on the implementation of the switching valve and the connecting ports which are switched through—is formed, for example, by the volumes of the following components (or a part thereof): pump head, mixer, connecting capillaries, sample loop, switching valve, metering device. In the case of HPG, the GDV—depending on the implementation of the switching valve and the connecting ports which are switched through—is formed, for example, by the volumes of the following components (or a part thereof): T-part, mixer, connecting capillaries, sample loop, switching valve, metering device. On the other hand, the washing-out volume must also be considered, which results due to the flow properties of the components.
If a chromatographic method was developed on a specific HPLC system, it usually cannot be transferred to another system without problems. The reproduction of a published method is also just as difficult. One cause of this is the different GDVs, which are accompanied by a shift and/or spreading of the retention times.
Furthermore, the mixture irregularity is also dependent on the GDV. The higher the GDV, the better the various solvents are mixed and the less the mixture irregularity.
The requirements for the GDV and the remaining mixture irregularity are also strongly dependent on the application and the detector type used. Non-optical detectors, such as MS, ELSD, or CAD, are typically insensitive to mixture irregularities, since these do not generate signal variations due to differing detection sensitivities to the individual solvent components. A mixer having small GDV with moderate mixer performance would therefore be acceptable in such a case. The situation appears very different, however, upon the use of a UV detector, above all at low wavelengths. Extremely small variations of the solvent composition have effects here in visible variations in the baseline. This makes it more difficult to determine the material concentration from the detector signal. A mixer having the highest possible mixing efficiency with correspondingly greater GDV would be advantageous in this case.
An unnecessarily large GDV is also advantageous, since the analysis, washing, and equilibration phases would also be increased unnecessarily.
If a method is transferred to another HPLC system, which has deviating GDVs, an offset time (tgdv=Vgdv/flow rate) could be calculated and the gradient forming could be started delayed or early accordingly.
However, problems with the irregularity cannot be completely remedied in this way. For this purpose, the mixer is normally manually replaced with another mixer. Of course, automation of such a replacement by means of a switching valve would also be conceivable. This solution would be cumbersome because of the size of the mixers and additionally costly, however.